FIG. 7.

Knockdown of PP2A-B56α increases S62- and T58-phosphorylated c-Myc as well as c-Myc-driven transcription. (A) c-Myc S62 and T58 phosphorylation increases upon PP2A-B56α knockdown. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-Myc-HA-WT, and 1.5 μg of pSUPER-shRNA expression vector [empty (−) or targeted to B56α, B56β, B56γ, B56δ, or B56ɛ, as indicated]. Whole-cell lysates were collected at 72 h posttransfection. Samples were normalized for β-gal activity and analyzed by Western blots (WB) simultaneously probed with anti-HA.11 for total c-Myc and either anti-T58phospho for T58 phosphorylation or anti-S62phospho for S62 phosphorylation. Anti-HA.11 was detected with secondary anti-mouse Alexa Fluor 680, and anti-T58phospho and anti-S62phospho were detected with secondary anti-rabbit IRDye800. The Western blots shown are representative of S62 phosphorylation, T58 phosphorylation, and total c-Myc protein levels from three separate experiments. (B) S62- and T58-phosphorylated c-Myc is enriched upon B56α knockdown. S62 or T58 phosphorylation levels along with total c-Myc levels, from simultaneously probed Western blots, were quantitated using LI-COR software (see “Western blotting and quantitation” in Materials and Methods). Ratios of S62-phosphorylation/total c-Myc and T58-phosphorylation/total c-Myc were calculated. Average ratios with error bars from the experiment shown in panel A and two repeat experiments were graphed relative to control (−) levels, and statistically significant differences are indicated (*). (C) B56α knockdown selectively increases c-Myc-driven transcription. HEK-293 cells were cotransfected with 50 ng CMV-βgal, 0.5 μg CMV-HA-c-Myc, and 10 ng of either E2F2-Luc or E2F2(-E-box)-Luc, along with 1.5 μg pSUPER-shRNA expression vector [empty (−) or targeted to B56α B56β, B56γ, B56δ, or B56ɛ, as indicated]. Whole-cell lysates were collected at 72 h posttransfection. c-Myc protein levels are shown by Western analysis with anti-HA.11. Luciferase activity was measured and corrected for transfection efficiency based on β-gal activity. Average corrected luciferase activity from three separate experiments was graphed with error bars, and statistically significant differences are indicated (*). (D) Knockdown of B56α increases endogenous E2F2 mRNA levels. HEK-293 cells were cotransfected with 50 ng CMV-βgal and 3 μg pSUPER-empty, pSUPER-B56α, or pSUPER-PP2A-A under 10% FBS conditions for 24 h and then starved in 0.2% FBS for 48 h. Cells exhibiting transfection efficiencies by β-gal assay within 5% of each other were used for RT-PCR analysis of endogenous E2F2, c-myc, and GAPDH mRNA levels.